(T1130-05-25) Hyper-Oxygenating Respiratoid Biosystem for Cell Transplantation

Purpose: Cell transplantation emerges as a prospective therapeutic strategy for addressing diseases associated with hormone or protein deficiencies. Despite notable advancements in this field, several challenges persist. A fundamental hurdle in cell transplantation is the challenge of providing sufficient oxygen to meet the demands of cells within 3-dimensional constructs. To enhance the oxygenation of tissue constructs, various types of oxygen-releasing biomaterials (ORBs) have been developed to date. However, there are consistent challenges in the development of advanced ORBs that can address burst oxygen release and spontaneous oxygenation issues. Also, in the oxygen decomposition process, byproducts referred to as reactive oxygen species (ROS) not only disrupt cellular pH and metabolic reactions but also induce cellular damage. Therefore, we try to overcome these drawbacks of the existing ORBs by spontaneously and consistently supplying oxygen to cells. To this end, we employed a chloroplast, which plays a pivotal role in photosynthesis by releasing oxygen from water. However, the isolated chloroplasts remain structurally intact for only a few hours in a test-tube, and photosynthetic activity is typically lost within 30 min to half a day. To overcome these constraints, we focused on the chloroplast proteins engaging with the translocon on the outer chloroplast membrane complex (TOC). In this context, we hypothesized that incorporating a chloroplast-transit-peptide (CTP) could potentially improve the structural instability of isolated chloroplasts through its affixation to the outer membrane. As a proof of concept, we introduced a ‘respiratoid’ biosystem utilizing isolated chloroplasts, CTP, and alginate polymer to offer a novel approach to spontaneous oxygen generation.

Methods: First, we confirmed the anchoring ability of CTP to chloroplast’s outer membrane through confocal microscopy. Next, we measured the photosynthetic activity of the isolated chloroplast treated with the CTP via chlorophyll fluorescence. The oxygenation of the chloroplast treated with the CTP was measured by SDR SensorDish® under hypoxic condition (1% pO2). We isolated photosystem II (PS II) from chloroplasts and confirmed the augment in protein expression of the oxygen-evolving complex with CTP treatment using SDS-PAGE analysis. We also performed RNA-seq and PCR to uncover the mechanism of CTP related to the improvement of chloroplast’s oxygenation. Alginate and CTP were conjugated via a carbodiimide coupling reaction to form an alginate-CTP conjugation polymer (Al-CTP). The conjugation of Al-CTP was characterized by 1H NMR spectroscopy and FT-IR analysis. To implement Respiratoid biosystem, Al-CTP is mixed in a 1:1 volume ratio with chloroplasts isolated from Spinacia oleracea, and pancreatic islets isolated from rats are encapsulated using an encapsulator. We tested the viability and functionality of encapsulated islets in the respiratoid biosystem under hypoxic condition by Live/Dead staining, CCK-8, insulin ELISA assay, and immunostaining. Finally, we subcutaneously and intraperitoneally transplanted the respiratoid biosystem to type 1 diabetic mouse model for long-term. We performed histological analysis of the retrieved repiratoid biosystem after 50 and 100 days.

Results: Through conjugation of CTP with alginate, we achieve stabilization of chloroplast structure. Strategically anchored to the outer chloroplast membrane, CTP not only ensures structural integrity but also upregulates key photosynthesis-associated genes and proteins. This biosystem demonstrates exceptional efficacy in spontaneously generating oxygen, particularly under hypoxic conditions (~1% pO2). In an application, pancreatic islets encapsulated within the respiratoid biosystem subcutaneouslly and intraperitoneally implanted in diabetic mice maintain normal glucose levels effectively. Insulin secretion persists for 100 days post-xenotransplantation without the need for immunosuppressant administration, highlighting the reliance on the respiratoid biosystem's oxygen supply and structural stability.

Conclusion: Our study demonstrates the respiratoid biosystem as a transformative platform in tissue engineering, offering a nature-inspired solution to the critical challenge of spontaneous oxygen supply. Overall, our respiratoid biosystem presents a novel approach that holds promise for advancing the field of oxygen-releasing biomaterials, particularly in the context of prolonged cellular support and potential applications in cell therapies.


a. Cumulated chlorophyll released from the destroyed chloroplasts for 14 days assessed by measuring absorbance 430 nm.
b. Comparison of oxygen levels of each chloroplast without light exposure at each time point under hypoxic conditions (1% pO2).


a. SDS-PAGE profile of each PSII complex at 14 days after isolation.
b. Gene Ontology (GO) analysis of mRNA-sequencing (mRNA-seq) quantifications.


a. Representative immunofluorescence images of islets after 20 h incubation under normoxic or hypoxic conditions.
b. Non-fasting blood glucose levels of the STZ-induced diabetic recipients after intraperitoneal xenotransplantation with respiratoid biosystem.